Integrins and other cell surface adhesion receptors regulate cell migration and invasion by mediating interactions between the extracellular matrix (ECM) and the actin cytoskeleton. Integrin- cytoskeleton interactions are highly regulated both temporally and spatially in migrating cells. The long term goal of our research is to identify and characterize the molecules that regulate integrin-cytoskeleton interactions during migration. Our previous studies identified the calcium-dependent protease calpain as a regulator of these interactions. Inhibition of calpain activity suppresses cell migration and invasiveness. The means by which calpain integrates with other cellular pathways to regulate cell migration remain largely unknown, and will be investigated in specific aims I and II. To identify other proteins that modulate cell migration we have employed an expression cloning scheme. Preliminary studies have identified candidate regulators, including a protein with sequence homology to the protein kinase C-binding protein RACK1. To characterize the molecular mechanisms that regulate integrin- cytoskeletal interactions during migration we propose the following Specific aims: I. Characterize the mechanisms that regulate the temporal and spatial distribution of mu-calpain in migrating cells. The adhesive and signaling pathways that modulate the intracellular distribution of mu-calpain during migration will be studied in live cells using a mu-calpain-GFP fusion protein. II. Characterize the relationship between mu- calpain function and integrin-cytoskeletal interactions. Using integrin mutants with well characterized binding to the cytoskeletal proteins talin or filamin, the effects of altered calpain activity on integrin-mediated migration and signal transduction will be examined. III. Identify and characterize novel proteins that regulate integrin-cytoskeletal interactions during cell migration. Expression cloning will be used to identify additional proteins that regulate integrin-cytoskeletal interactions during migration. The RACK1 homolog identified in early rounds of screening will be characterized. These studies will enhance our understanding of the molecular mechanisms that regulate cell migration and provide information relevant to pathological processes such as tumor invasion and metastasis.